The present invention relates to biological engineering, and more particularly, to apparatus for performing electro cell fusion on a large scale basis.
The electro cell fusion process is generally done in several phases. In the first phase, the cells are brought close to each other to form pearl chains by exposing the cells to an alternating electric field. In the second phase, the cells which are in contact with each other are exposed for a brief moment to an alternating field of higher amplitude than the initial alternating electric field. The cells are pushed against each other and flatten out in the area of contact. In the third phase, cell fusion is initiated by one or more short, unidirectional pulses of high voltage. Under the correct conditions, pores in the cell membranes will open up and adjacent cells will fuse with each other. The resulting hybrid cell contains the genetic information of both the original cells. If the voltage is too high or the pulses too long, cell damage can occur and a non-viable hybrid results. There are particular parameters for optimal fusion yield for different cell types. In the fourth phase, the alignment alternating electric field is reapplied to maintain mechanical confinement and to aid in rounding off the fused cells.
Two physical processes are involved in the four phases of electro cell fusion. Dielectrophoresis governs the movement of the cells, i.e. alignment, compression and post fusion. Dielectric breakdown governs the actual fusion event.
Heretofore, electro cell fusion has been performed on a small scale utilizing an apparatus consisting of wires or thin metal plates on a microscope slide. A droplet of a fluid containing the live cells in suspension is deposited on this type of apparatus so that it bridges the two wires or metal plates. Suitable electric currents are then applied to the wires or plates. The droplet of cell suspension fluid is held in position by capillary action and surface tension. The volume of fluid which can be treated with such a device is typically on the order of one micro-liter. Examples of microscope slide type electro cell fusion devices are commercially available from GCA Corporation of Chicago, Ill. and D.E.P. Systems, Inc. of Metamora, Miss.
Another prior electro cell fusion device maintains the cell suspension fluid in a closed loop to permit repetitive sterile injection of fresh cells, with fused cells exiting the opposite end. One such device is available from D.E.P. Systems, Inc. and another such device is illustrated in FIG. 6 of U.S. Pat. No. 4,441,972.
It would be desirable to provide an apparatus for performing electro cell fusion on much larger fluid volumes, containing much larger number of cells to be fused, than has been possible with prior such devices. Such a large volume cell fusion apparatus would permit large scale production of hybrid cells for manufacturing monoclonal antibodies, among other uses.
U.S. Pat. No. 4,441,972 referred to above also discloses a cell sorting/electro cell fusion apparatus which is supposed to handle a large number of cells. It includes a very flat disk-shaped chamber defined between upper and lower electrode plates. The lower electrode has a plurality of concentric V-shaped grooves cut in in its upper surface. A cell suspension fluid inlet extends through the center of the lower electrode and an outlet extends through the periphery of the lower electrode. This apparatus depends upon surface effects of the grooved electrode in performing electro cell fusion. The mechanical structure is complex and requires precise parallel alignment of the electrode plates. It is not feasible to produce large volumes of viable hybrid cells with this device.